Normal human cells in vitro exhibit a stringent limitation of division capacity in contrast to tumor-derived and virus-, carcinogen- or irradiation-transformed cells that can divide indefinitely (immortal). We do not yet understand the mechanisms that limit the division potential of normal human cells or the changes occurring to yield immortal cells. However, from cell hybrid studies we have found that the immortal phenotype results from recessive changes in normal cell growth control. We have exploited this fact to separate twenty four different immortal cell lines into four implementation groups for indefinite division. This indicates that there are at least four paths to immortality and that at least four genes or sets of genes are involved in normal cell growth control. We have also found that normal senescent human cells express a protein inhibitor of DNA synthesis and contain a high abundance of mRNAs that inhibit DNA synthesis, neither of which is present in young proliferating cells. Our current hypothesis is that expression of this protein is the end- point of a genetic program that results in cellular senescence. Now that we have assigned immortal cell lines to specific groups we can proceed to determine what changes have occurred that are common to cell lines within a complementation group that could be involved in immortalization. Cytogenetic analysis combined with the use of restriction fragment length polymorphisms have allowed for the identification of specific genetic changes associated with, for example, certain types of tumors, cystic fibrosis and Duchenne muscular dystrophy. We plan to take a similar approach to identify chromosomal changes that are common to immortal cell lines within a complementation group. We can then determine whether these chromosomes are involved in immortalization by hybrid or microcell fusion studies. We will also analyze cell lines within the different implementation groups to determine whether they have become immortal because of changes in expression of the protein inhihitor of DNA synthesis that is produced in senescent cells. The results from these studies should provide further insight into changes at the genetic and molecular level that are involved in immortalization of human cells and thereby to the genes or sets of genes involved in cellular senescence.